A time-resolved multi-omics atlas of Acanthamoeba castellanii encystment

Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence of the bacteria they harbor. Here, we mapped the global molecular changes that occur in the facultatively pathogenic amoeba Acanthamoeba castellanii during the early steps of the poorly understood process of encystment. By performing transcriptomic, proteomic, and phosphoproteomic experiments during encystment, we identified more than 150,000 previously undescribed transcripts and thousands of protein sequences absent from the reference genome. These results provide molecular details to the regulation of expected biological processes, such as cell proliferation shutdown, and reveal new insights such as a rapid phospho-regulation of sites involved in cytoskeleton remodeling and translation regulation. This work constitutes the first time-resolved molecular atlas of an encysting organism and a useful resource for further investigation of amoebae encystment to allow for a better control of pathogenic amoebae.

While most parts of the manuscript are very well written, the first part of the introduction is, to my opinion, somehow clumsy and unnecessarily exaggerating the medical relevance of Acanthamoeba. lines 38-39: firstly, not all FLA belong to the Amoebozoa, and secondly, the FLA are surely not the most important human pathogens. Here, Entamoeba histolytica has to be mentioned.
lines 41-42: this is too anthropomorphic, they do not prevent their eradication, in fact, FLA normally do not have contact to the human immune system or biocides; all this is purely accidental lines 43-51: I suggest to focus here on Acanthamoeba or at least on the Amoebozoa; Naegleria is not related at all and also its cysts are very different. lines 52-59: I suggest to rephrase, to make it less dramatic and easier to understand. The reason why they may function as training grounds mainly is that they are phagocytotic eukaryotic cells and thus similar to human immune cells, e.g. macrophages.
lines 87-88: correct English; this would mean that the genome is easys to encyst. line 90: analyses lines 110-116: in the literature there have been several reports on Acanthamoeba producing immature cysts and so-called pseudocysts: has this been taken into account? I acknowledge that the authors used calcofluor staining, which likely binds best to mature cysts, but was the cyst wall checked microcopically (if doubled)?  The molecular mechanism of cyst formation is unclear yet in Acanthamoeba. The authors tried very vast effort to discover this secret. Up to date, most researchers studied on similar approach on Acanthamoeba faced a big problem that large portion of Acanthamoeba genes annotated as hypothetical protein. Even they showed high frequency during encystation, it is hard to investigate further function of the gene. In this paper, authors didn't mention about hypothetical protein. Please explain that how much portin of genes annotated as hypothetical protein, and by how you overcome this problem to do further research.
The authors use RNAseq, proteomics, and phosphoproteomics to characterize the early stages of encystation by Acanthamoeba castellanii, a free-living protist that causes keratitis and amoebic encephalitis. Their methods are vigorous, and their presentation is clear and does not over-interpret their data.
Their major findings include: 1) identification of many many more protein-encoding genes than previously annotated by the initial genome project.
2) demonstration of 18 patterns of gene expression during the transition from trophozoite to cyst, which suggests gene expression is coregulated in predictable and unpredictable ways.
3) demonstration that changes in gene expression and protein phosphorylation are fast, while changes in protein abundance is slow.

4) a series of stories, mostly included in the Discussion, suggesting transcription factors, kinases/phosphatases, actin-associated proteins, proteosomes, autosomes, and cell wall proteins/enzymes that are important for the encystation process.
This is an enormous amount of work that will be useful for all those investigating this poorly characterized protist. For example, the fasta file of proteins deposited in Zenodo makes it possible to correct protein predictions for AmoebaDB that are wrong, because of incorrect sequence assemblies or incorrect exon prediction.
We thank the reviewer for his thorough evaluation of our manuscript and for his positive comments.
Limitations to this work are:

1) 8 hour time point is the beginning of encystation that is not completed until 72 hours, so that many aspects of encystation are missed.
We agree with the comment of reviewer 1 that encystation is generally not completed until 72h. However, as stated lines 21-22 or 100-103 of the manuscript, we chose to focus on the early steps of encystment to study molecular regulations that underly -and may be responsible for-A. castellanii encystment.
We inserted the following sentences to explain the choice of our time-points: "We considered the 1h time-point because it precedes the maximum expression of the trehalose synthase gene observed at 2h in encysting cells 24 . We opted for 4h and 8h, as Hirukawa et al. did not detect the cyst-specific protein CSP21, a marker of A. castellanii encystment, at these time-points 25 ." (lines 123 -126)

2) it would be great if data was placed in AmoebaDB, where it is much easier to access individual genes.
We thank the reviewer for this suggestion. Transcriptome and proteome were submitted to AmoebaDB. We are still in the process of making them available to the community, in the meantime they are listed in the list of data sets that the AmoebaDB team is in the process of loading (https://amoebadb.org/amoeba/app/static-content/dataInprogress.html see screenshot below).

Reviewer #2 (Remarks to the Author):
With this manuscript, the authors provide the first multi-omics atlas of Acanthamoeba encystment. The study was well-designed and offers a sound and valuable basis not only for all molecular studies on Acanthamoeba but also for studying encystment in other protists. The manuscript is very well written in most parts and is overall clear and easy to follow. To my opinion, it is suitable for publication after minor revision.
We thank the reviewer for this positive comment.

Specific comments:
While most parts of the manuscript are very well written, the first part of the introduction is, to my opinion, somehow clumsy and unnecessarily exaggerating the medical relevance of Acanthamoeba.
We have modified this part to improve readability (see below).
lines 38-39: firstly, not all FLA belong to the Amoebozoa, and secondly, the FLA are surely not the most important human pathogens. Here, Entamoeba histolytica has to be mentioned.
Thank you. We have reworded these lines (37 to 40) to state: "Among protist, some free-living and parasitic amoebae, such as Acanthamoeba castellanii and Entamoeba histolytica, can undergo encystment". Thank you for raising this point. We have made the sentence clearer to the following: "This ensures species sustainability by allowing them to survive in the environment due to cysts high resilience" (lines 40 -43).
lines 43-51: I suggest to focus here on Acanthamoeba or at least on the Amoebozoa; Naegleria is not related at all and also its cysts are very different.
Thank you for this suggestion. We have modified the manuscript accordingly to state: "For example, the genus Acanthamoeba was reported to cause fatal amoebic meningoencephalitis" (lines 47 -48).
lines 52-59: I suggest to rephrase, to make it less dramatic and easier to understand. The reason why they may function as training grounds mainly is that they are phagocytotic eukaryotic cells and thus similar to human immune cells, e.g. macrophages.
This part has been shortened as follows: "In addition, some bacteria have been shown to become more virulent and more resistant to biocides and antibiotics after their passage through amoebae." (lines 57-58).

lines 87-88: correct English; this would mean that the genome is easys to encyst.
The sentence has been reformulated to correct this error: "For these reasons, we investigated in this study the encystment of A. castellanii, a model eukaryote for cellular biology, as it readily undergoes encystment in starvation conditions and has an annotated genome available." (lines 88-90)

line 90: analyses
This error has been corrected (line 92).
lines 110-116: in the literature there have been several reports on Acanthamoeba producing immature cysts and so-called pseudocysts: has this been taken into account? I acknowledge that the authors used calcofluor staining, which likely binds best to mature cysts, but was the cyst wall checked microcopically (if doubled)?
Yes, Acanthamoeba does produce pseudocysts, and this is something we have taken into account. We always checked microscopically the state of the cysts before harvesting cells for further experiments, and we always observed the presence of cyst walls composed of two layers. This information has been included in the manuscript (lines 499 to 501).

line 194: processes
This error has been corrected (line 197).
line 264: how were these time points actually chosen? What was the rationale for choosing exactly these time points?
Thank you for this comment. To clarify, we have inserted the following sentences: "We considered the 1h time-point because it precedes the maximum expression of the trehalose synthase gene observed at 2h in encysting cells 24 . We opted for 4h and 8h, as Hirukawa et al. did not detect the cyst-specific protein CSP21, a marker of A. castellanii encystment, at these time-points 25 ." (lines 123 -126).

line 272: cells
This error has been corrected (line 275).

line 326: delete "be"
This error has been corrected (line 329).
line 483: The Neff strain is known to not encyst easily (Parasitol Res (2008) 102:1069-1072), were the cultures pre-treated/subcultured somehow? This would be important to know for the reader.
As shown on the Supplementary Fig. 1c, we agree with the reviewer that the Neff strain encysts slowly. No special pre-treatment nor subculture were used in addition to the protocol described (lines 489-515). We have generated several frozen stocks of A. castellanii. In that way, we never use amoebae subcultured for more than 20 times. Once they reach that point, we start from a new stock. This information has been included in the manuscript (lines 486-487).
lines 489-534: while most parts of the manuscript are very well written, this parts needs revision of English language, numerous grammar mistakes.
This part of the manuscript has been corrected (lines 489 to 540).

line 550: the 2 should be subscripted
This error has been corrected (line 555).

line 612: delete "of"
This error has been corrected (line 620).

line 638: Peptides
This error has been corrected (line 644).

line 653: vortexed and centrifuged
These errors have been corrected (line 659).

Reviewer #3 (Remarks to the Author):
The molecular mechanism of cyst formation is unclear yet in Acanthamoeba. The authors tried very vast effort to discover this secret. Up to date, most researchers studied on similar approach on Acanthamoeba faced a big problem that large portion of Acanthamoeba genes annotated as hypothetical protein. Even they showed high frequency during encystation, it is hard to investigate further function of the gene. In this paper, authors didn't mention about hypothetical protein. Please explain that how much portin of genes annotated as hypothetical protein, and by how you overcome this problem to do further research.
Reviewer #3 raises two important points regarding organisms with poorly annotated genomes such as A. castellanii: -genes annotated as hypothetical proteins are genes that are annotated from sequence similarity, but we do not have any evidence of their transcription/translation to functional proteins. In the Uniprot/TrEMBL proteome of A. castellanii (5th of May 2022), we found less than 20 proteins with evidence at protein level (field "Protein existence"), although this number is an underestimation because this species is not well annotated. Therefore, there are many hypothetical proteins. Our MS-based proteogenomics data set provides evidence of the presence of many proteins in A. castellanii during encysting conditions, therefore we focused our discussion on data at the protein level.
-genes with no functional annotation: many genes of A. castellanii do not have a known function. We retrieved functional annotation using eggnog-mapper (http://eggnogmapper.embl.de/) that transfers functional annotations from orthologs. This allowed the annotation of ~1900 protein sequences that were not in Uniprot/TrEMBL (5000+ proteins). All these annotations are available in the supplementary tables of the manuscript, and we believe that these are helpful when mining our data. To clarify this point in the manuscript, we have modified lines 212 -215 as follows: "With this custom database we identified 8,577 protein groups (proteins that cannot be distinguished by MS because identified by a common set of peptides) (Fig. 3a) among which 2,342 had no functional annotation retrieved using eggNOGmapper.".
Of course, hypothetical genes with unknown functions remain a crucial issue when studying organisms such as amoebas. Indeed, these are particularly difficult to engineer in vitro, and high-throughput screening through genetic manipulation remains out of the question. In our group, we are currently working on the silencing of a few targets that proved to be very challenging. In this context, generation of data sets such as the one we provide here help in reducing the number of uncharacterized genes to focus on by providing a list of genes that are expressed and regulated in a given biological context. Furthermore, knowing this biological context (here encystment), can aid in the design of future experiments for functional validation of these genes.